quantifying my gene expression in 5 different cell lines - (Oct/22/2007 )
I am still learning the nitty-gritty of performing a RTPCR...I usually run qRTPCR by the Taqman method, comparing gene expression levels between GFP and siRNA treated cells and look for downregulation. I use
RNAse P as the internal control for normalization and use transfected HEY cell line.
However, I m now confused as I am required to check for my gene's expression level in 5 different
cancer cell lines. Since i just have to quantitate the expression and not really comapre against anything,
I am a little lost about how shd I go about running the samples and calculating using excel sheet.
SHould i consider one of the cell line as baseline i.e 1 and check expression levels?
and what shd be the other controls?
not sure if I am putting across my question effectively...can anyone reply please?
Thanks for any help...
In Real-time PCR yo can't quantiate the expression level in your test sample without comparing it to your control (untreated). In your expt, the untreated GFP and SiRNA will be the control sample and treated GFP and SiRNA will be the test sample. Do amplify the RNAseP gene and your gene of interest in both control and test samples. Then using Ct method you can calc the gene expression in your test sample after normalisation.